The overall goal of the project is to determine the mechanisms of ethanol toxicity to the pancreas which mediate pancreatitis, a poorly understood and treated disorder with alcohol abuse as a major cause. The work proposed in the application is designed to determine the pathways of ethanol metabolism in pancreatic acinar cells and the mechanisms whereby ethanol metabolites mediate pancreatic acinar cell injury. We hypothesize that pancreatic acinar cell produces non-oxidative (fatty acid ethyl esters, FAEEs) and oxidative (acetaldehyde and acetate) ethanol metabolites. The ethanol metabolites, in turn, activate transcription factors, such as NF-kappaB and AP-1. The transcription factors activate the expression of cytokines that are responsible for inflammation, cell death, and fibrosis, the main characteristics of ethanol-induced pancreatitis. We propose the following specific aims: 1. Measure non-oxidative and oxidative ethanol metabolism in dispersed rat pancreatic acini and hepatocytes by measuring the activities of FAEE synthase, alcohol dehydrogenase, aldehyde dehydrogenase, the formation of FAEEs, acetaldehyde, and acetate in pancreatic acini incubated with ethanol for different times; and the intracellular localization (cytosol, microsomes) of the ethanol metabolism. 2. Determine the effects of ethanol, FAEEs, acetaldehyde, and acetate on the activation of transcription factors NF- kappaB and AP-1 in dispersed pancreatic acini 3. Determine the effects of ethanol. FAEEs, acetaldehyde and acetate on the expression of cytokines TNFalpha, IL-1, IL-6, and TGFbeta in dispersed pancreatic acini. 4. Measure non-oxidative and oxidative ethanol metabolism in pancreatic tissue from rats fed ethanol by intragastric infusion for different period of time. To accomplish these goals, we will perform the following measurements on rat pancreatic acinar cells and hepatocytes, measurements of the activities of alcohol dehydrogenase, aldehyde dehydrogenase, FAEE synthase in crude cell homogenate and cell fractions using enzyme assays; FAEE and acetate accumulation in intact cells using thin layer and ion-exchange chromatography; transcription factor activation using electromobility shift assays: expression of cytokines/chemokines using quantitative RT-PCR. The result of the experiments described in this application will be the identification of key mechanisms of ethanol toxicity for pancreatic acinar cells that can be targets for therapeutic intervention.